Detecting T cell receptors involved in immune responses from single repertoire snapshots.

Hypervariable T cell receptors (TCRs) play a key role in adaptive immunity, recognizing a vast diversity of pathogen-derived antigens. Our ability to extract clinically relevant information from large high-throughput sequencing of TCR repertoires (RepSeq) data is limited, because little is known about TCR-disease associations. We present Antigen-specific Lymphocyte Identification by Clustering of Expanded sequences (ALICE), a statistical approach that identifies TCR sequences actively involved in current immune responses from a single RepSeq sample and apply it to repertoires of patients with a variety of disorders - patients with autoimmune disease (ankylosing spondylitis [AS]), under cancer immunotherapy, or subject to an acute infection (live yellow fever [YF] vaccine). We validate the method with independent assays. ALICE requires no longitudinal data collection nor large cohorts, and it is directly applicable to most RepSeq datasets. Its results facilitate the identification of TCR variants associated with diseases and conditions, which can be used for diagnostics and rational vaccine design.

The Absence of Retroelement Activity Is Characteristic for Childhood Acute Leukemias and Adult Acute Lymphoblastic Leukemia.

Retroelements (RE) have been proposed as important players in cancerogenesis. Different cancer types are characterized by a different level of tumor-specific RE insertions. In previous studies, small cohorts of hematological malignancies, such as acute myeloid leukemia, multiple myeloma, and chronic lymphocytic leukemia have been characterized by a low level of RE insertional activity. Acute lymphoblastic leukemia (ALL) in adults and childhood acute leukemias have not been studied in this context. We performed a search for new RE insertions (Alu and L1) in 44 childhood ALL, 14 childhood acute myeloid leukemia, and 14 adult ALL samples using a highly sensitive NGS-based approach. First, we evaluated the method sensitivity revealing the 1% detection threshold for the proportion of cells with specific RE insertion. Following this result, we did not identify new tumor-specific RE insertions in the tested cohort of acute leukemia samples at the established level of sensitivity. Additionally, we analyzed the transcription levels of active L1 copies and found them increased. Thus, the increased transcription of active L1 copies is not sufficient for overt elevation of L1 retrotranspositional activity in leukemia.

Precise tracking of vaccine-responding T cell clones reveals convergent and personalized response in identical twins.

T cell receptor (TCR) repertoire data contain information about infections that could be used in disease diagnostics and vaccine development, but extracting that information remains a major challenge. Here we developed a statistical framework to detect TCR clone proliferation and contraction from longitudinal repertoire data. We applied this framework to data from three pairs of identical twins immunized with the yellow fever vaccine. We identified 600 to 1,700 responding TCRs in each donor and validated them using three independent assays. While the responding TCRs were mostly private, albeit with higher overlap between twins, they could be well-predicted using a classifier based on sequence similarity. Our method can also be applied to samples obtained postinfection, making it suitable for systematic discovery of new infection-specific TCRs in the clinic.

High-throughput sequencing of T-cell receptor alpha chain clonal rearrangements at the DNA level in lymphoid malignancies.

Rearrangements of T- and B-cell receptor (TCR and BCR) genes are useful markers for clonality assessment as well as for minimal residual disease (MRD) monitoring during the treatment of haematological malignancies. Currently, rearrangements of three out of four TCR and all BCR loci are used for this purpose. The fourth TCR gene, TRA, has not been used so far due to the lack of a method for its rearrangement detection in genomic DNA. Here we propose the first high-throughput sequencing based method for the identification of clonal TRA gene rearrangements at the DNA level. The method is based on target amplification of the rearranged TRA locus using an advanced multiplex polymerase chain reaction system and high-throughput sequencing, and has been tested on DNA samples from peripheral blood of healthy donors. Combinations of all functional V- and J-segments were detected, indicating the high sensitivity of the method. Additionally, we identified clonal TRA rearrangements in 57 out of 112 tested DNA samples of patients with various T-lineage lymphoproliferative disorders. The method fills the existing gap in utilizing the TRA gene for a wide range of studies, including clonality assessment, MRD monitoring and clonal evolution analysis in different lymphoid malignancies.

Distinctive properties of identical twins' TCR repertoires revealed by high-throughput sequencing.

Adaptive immunity in humans is provided by hypervariable Ig-like molecules on the surface of B and T cells. The final set of these molecules in each organism is formed under the influence of two forces: individual genetic traits and the environment, which includes the diverse spectra of alien and self-antigens. Here we assess the impact of individual genetic factors on the formation of the adaptive immunity by analyzing the T-cell receptor (TCR) repertoires of three pairs of monozygous twins by next-generation sequencing. Surprisingly, we found that an overlap between the TCR repertoires of monozygous twins is similar to an overlap between the TCR repertoires of nonrelated individuals. However, the number of identical complementary determining region 3 sequences in two individuals is significantly increased for twin pairs in the fraction of highly abundant TCR molecules, which is enriched by the antigen-experienced T cells. We found that the initial recruitment of particular TCR V genes for recombination and subsequent selection in the thymus is strictly determined by individual genetic factors. J genes of TCRs are selected randomly for recombination; however, the subsequent selection in the thymus gives preference to some α but not ß J segments. These findings provide a deeper insight into the mechanism of TCR repertoire generation.

Novel bimodal TRBD1-TRBD2 rearrangements with dual or absent D-region contribute to TRB V-(D)-J combinatorial diversity.

T-cell receptor (TR) diversity of the variable domains is generated by recombination of both the alpha (TRA) and beta (TRB) chains. The textbook process of TRB chain production starts with TRBD and TRBJ gene rearrangement, followed by the rearrangement of a TRBV gene to the partially rearranged D-J gene. Unsuccessful V-D-J TRB rearrangements lead to apoptosis of the cell. Here, we performed deep sequencing of the poorly explored pool of partial TRBD1-TRBD2 rearrangements in T-cell genomic DNA. We reconstructed full repertoires of human partial TRBD1-TRBD2 rearrangements using novel sequencing and validated them by detecting V-D-J recombination-specific byproducts: excision circles containing the recombination signal (RS) joint 5'D2-RS - 3'D1-RS. Identified rearrangements were in compliance with the classical 12/23 rule, common for humans, rats, and mice and contained typical V-D-J recombination footprints. Interestingly, we detected a bimodal distribution of D-D junctions indicating two active recombination sites producing long and short D-D rearrangements. Long TRB D-D rearrangements with two D-regions are coding joints D1-D2 remaining classically on the chromosome. The short TRB D-D rearrangements with no D-region are signal joints, the coding joint D1-D2 being excised from the chromosome. They both contribute to the TRB V-(D)-J combinatorial diversity. Indeed, short D-D rearrangements may be followed by direct V-J2 recombination. Long D-D rearrangements may recombine further with J2 and V genes forming partial D1-D2-J2 and then complete V-D1-D2-J2 rearrangement. Productive TRB V-D1-D2-J2 chains are present and expressed in thousands of clones of human antigen-experienced memory T cells proving their capacity for antigen recognition and actual participation in the immune response.

The use of non-functional clonotypes as a natural calibrator for quantitative bias correction in adaptive immune receptor repertoire profiling.

High-throughput sequencing of adaptive immune receptor repertoires is a valuable tool for receiving insights in adaptive immunity studies. Several powerful TCR/BCR repertoire reconstruction and analysis methods have been developed in the past decade. However, detecting and correcting the discrepancy between real and experimentally observed lymphocyte clone frequencies are still challenging. Here, we discovered a hallmark anomaly in the ratio between read count and clone count-based frequencies of non-functional clonotypes in multiplex PCR-based immune repertoires. Calculating this anomaly, we formulated a quantitative measure of V- and J-genes frequency bias driven by multiplex PCR during library preparation called Over Amplification Rate (OAR). Based on the OAR concept, we developed an original software for multiplex PCR-specific bias evaluation and correction named iROAR: immune Repertoire Over Amplification Removal (https://github.com/smiranast/iROAR). The iROAR algorithm was successfully tested on previously published TCR repertoires obtained using both 5' RACE (Rapid Amplification of cDNA Ends)-based and multiplex PCR-based approaches and compared with a biological spike-in-based method for PCR bias evaluation. The developed approach can increase the accuracy and consistency of repertoires reconstructed by different methods making them more applicable for comparative analysis.

Effect of High-Pressure Torsion and Annealing on the Structure, Phase Composition, and Microhardness of the Ti-18Zr-15Nb (at. %) Alloy.

The Ti-18Zr-15Nb shape memory alloys are a new material for medical implants. The regularities of phase transformations during heating of this alloy in the coarse-grained quenched state and the nanostructured state after high-pressure torsion have been studied. The specimens in quenched state (Q) and HPT state were annealed at 300-550 °C for 0.5, 3, and 12 h. The α-phase formation in Ti-18Zr-15Nb alloy occurs by C-shaped kinetics with a pronounced peak near 400-450 °C for Q state and near 350-450 °C for HPT state, and stops or slows down at higher and lower annealing temperatures. The formation of a nanostructured state in the Ti-18Zr-15Nb alloy as a result of HPT suppresses the ß→ω phase transformation during low-temperature annealing (300-350 °C), but activates the ß→α phase transformation. In the Q-state the α-phase during annealing at 450-500 °C is formed in the form of plates with a length of tens of microns. The α-phase formed during annealing of nanostructured specimens has the appearance of nanosized particle-grains of predominantly equiaxed shape, distributed between the nanograins of ß-phase. The changes in microhardness during annealing of Q-specimens correlate with changes in phase composition during aging.

Inactivated tick-borne encephalitis vaccine elicits several overlapping waves of T cell response.

The development and implementation of vaccines have been growing exponentially, remaining one of the major successes of healthcare over the last century. Nowadays, active regular immunizations prevent epidemics of many viral diseases, including tick-borne encephalitis (TBE). Along with the generation of virus-specific antibodies, a highly effective vaccine should induce T cell responses providing long-term immune defense. In this study, we performed longitudinal high-throughput T cell receptor (TCR) sequencing to characterize changes in individual T cell repertoires of 11 donors immunized with an inactivated TBE vaccine. After two-step immunization, we found significant clonal expansion of both CD4+ and CD8+ T cells, ranging from 302 to 1706 vaccine-associated TCRß clonotypes in different donors. We detected several waves of T cell clonal expansion generated by distinct groups of vaccine-responding clones. Both CD4+ and CD8+ vaccine-responding T cell clones formed 17 motifs in TCRß sequences shared by donors with identical HLA alleles. Our results indicate that TBE vaccination leads to a robust T cell response due to the production of a variety of T cell clones with a memory phenotype, which recognize a large set of epitopes.

Microwave Discharges in Liquid Hydrocarbons: Physical and Chemical Characterization.

Microwave discharges in dielectric liquids are a relatively new area of plasma physics and plasma application. This review cumulates results on microwave discharges in wide classes of liquid hydrocarbons (alkanes, cyclic and aromatic hydrocarbons). Methods of microwave plasma generation, composition of gas products and characteristics of solid carbonaceous products are described. Physical and chemical characteristics of discharge are analyzed on the basis of plasma diagnostics and 0D, 1D and 2D simulation.

Longitudinal high-throughput TCR repertoire profiling reveals the dynamics of T-cell memory formation after mild COVID-19 infection.

COVID-19 is a global pandemic caused by the SARS-CoV-2 coronavirus. T cells play a key role in the adaptive antiviral immune response by killing infected cells and facilitating the selection of virus-specific antibodies. However, neither the dynamics and cross-reactivity of the SARS-CoV-2-specific T-cell response nor the diversity of resulting immune memory is well understood. In this study, we use longitudinal high-throughput T-cell receptor (TCR) sequencing to track changes in the T-cell repertoire following two mild cases of COVID-19. In both donors, we identified CD4+ and CD8+ T-cell clones with transient clonal expansion after infection. We describe characteristic motifs in TCR sequences of COVID-19-reactive clones and show preferential occurrence of these motifs in publicly available large dataset of repertoires from COVID-19 patients. We show that in both donors, the majority of infection-reactive clonotypes acquire memory phenotypes. Certain T-cell clones were detected in the memory fraction at the pre-infection time point, suggesting participation of pre-existing cross-reactive memory T cells in the immune response to SARS-CoV-2.

Identification of Disease-associated Traits and Clonotypes in the T Cell Receptor Repertoire of Monozygotic Twins Affected by Inflammatory Bowel Diseases.

BACKGROUND AND AIMS: Intestinal inflammation in inflammatory bowel diseases [IBD] is thought to be T cell mediated and therefore dependent on the interaction between the T cell receptor [TCR] and human leukocyte antigen [HLA] proteins expressed on antigen presenting cells. The collection of all TCRs in one individual, known as the TCR repertoire, is characterised by enormous diversity and inter-individual variability. It was shown that healthy monozygotic [MZ] twins are more similar in their TCR repertoire than unrelated individuals. Therefore MZ twins, concordant or discordant for IBD, may be useful to identify disease-related and non-genetic factors in the TCR repertoire which could potentially be used as disease biomarkers. METHODS: Employing unique molecular barcoding that can distinguish between polymerase chain reaction [PCR] artefacts and true sequence variation, we performed deep TCRα and TCRß repertoire profiling of the peripheral blood of 28 MZ twin pairs from Denmark and Germany, 24 of whom were discordant and four concordant for IBD. RESULTS: We observed disease- and smoking-associated traits such as sharing, diversity and abundance of specific clonotypes in the TCR repertoire of IBD patients, and particularly in patients with active disease, compared with their healthy twins. CONCLUSIONS: Our findings identified TCR repertoire features specific for smokers and IBD patients, particularly when signs of disease activity were present. These findings are a first step towards the application of TCR repertoire analyses as a valuable tool to characterise inflammatory bowel diseases and to identify potential biomarkers and true disease causes.

Primary and secondary anti-viral response captured by the dynamics and phenotype of individual T cell clones.

The diverse repertoire of T-cell receptors (TCR) plays a key role in the adaptive immune response to infections. Using TCR alpha and beta repertoire sequencing for T-cell subsets, as well as single-cell RNAseq and TCRseq, we track the concentrations and phenotypes of individual T-cell clones in response to primary and secondary yellow fever immunization - the model for acute infection in humans - showing their large diversity. We confirm the secondary response is an order of magnitude weaker, albeit ∼10 days faster than the primary one. Estimating the fraction of the T-cell response directed against the single immunodominant epitope, we identify the sequence features of TCRs that define the high precursor frequency of the two major TCR motifs specific for this particular epitope. We also show the consistency of clonal expansion dynamics between bulk alpha and beta repertoires, using a new methodology to reconstruct alpha-beta pairings from clonal trajectories.

SeqURE - a new copy-capture based method for sequencing of unknown Retroposition events.

BACKGROUND: Retroelements (REs) occupy a significant part of all eukaryotic genomes including humans. The majority of retroelements in the human genome are inactive and unable to retrotranspose. Dozens of active copies are repressed in most normal tissues by various cellular mechanisms. These copies can become active in normal germline and brain tissues or in cancer, leading to new retroposition events. The consequences of such events and their role in normal cell functioning and carcinogenesis are not yet fully understood. If new insertions occur in a small portion of cells they can be found only with the use of specific methods based on RE enrichment and high-throughput sequencing. The downside of the high sensitivity of such methods is the presence of various artifacts imitating real insertions, which in many cases cannot be validated due to lack of the initial template DNA. For this reason, adequate assessment of rare (< 1%) subclonal cancer specific RE insertions is complicated. RESULTS: Here we describe a new copy-capture technique which we implemented in a method called SeqURE for Sequencing Unknown of Retroposition Events that allows for efficient and reliable identification of new genomic RE insertions. The method is based on the capture of copies of target molecules (copy-capture), selective amplification and sequencing of genomic regions adjacent to active RE insertions from both sides. Importantly, the template genomic DNA remains intact and can be used for validation experiments. In addition, we applied a novel system for testing method sensitivity and precisely showed the ability of the developed method to reliably detect insertions present in 1 out of 100 cells and a substantial portion of insertions present in 1 out of 1000 cells. Using advantages of the method we showed the absence of somatic Alu insertions in colorectal cancer samples bearing tumor-specific L1HS insertions. CONCLUSIONS: This study presents the first description and implementation of the copy-capture technique and provides the first methodological basis for the quantitative assessment of RE insertions present in a small portion of cells.

Human trash ESTs--sequences from cDNA collection that are not aligned to genome assembly.

Expressed sequence tags (ESTs) represent 500-1000-bp-long sequences corresponding to mRNAs derived from different sources (cell lines, tissues, etc.). The human EST database contains over 8,000,000 sequences, with over 4,000,000,000 total nucleotides. RNA molecules are transcribed from a genomic DNA template; therefore, all ESTs should match corresponding genomes. Nevertheless, we have found in the human EST database approximately 11,000 ESTs not matching sequences in the human genome database. The presence of "trash" ESTs (TESTs) in the EST database could result from DNA or RNA contamination of the laboratory equipment, tissues, or cell lines. TESTs could also represent sequences from unidentified human genes or from species inhabiting the human body. Here, we attempt to identify the sources of human EST database contaminations. In particular, we discuss systematic contamination of the mammalian EST databases with sequences of plants.

Method for identification of condition-associated public antigen receptor sequences.

Diverse repertoires of hypervariable immunoglobulin receptors (TCR and BCR) recognize antigens in the adaptive immune system. The development of immunoglobulin receptor repertoire sequencing methods makes it possible to perform repertoire-wide disease association studies of antigen receptor sequences. We developed a statistical framework for associating receptors to disease from only a small cohort of patients, with no need for a control cohort. Our method successfully identifies previously validated Cytomegalovirus and type one diabetes responsive TCR[Formula: see text] sequences .

An advanced enrichment method for rare somatic retroelement insertions sequencing.

BACKGROUND: There is increasing evidence that the transpositional activity of retroelements (REs) is not limited to germ line cells, but often occurs in tumor and normal somatic cells. Somatic transpositions were found in several human tissues and are especially typical for the brain. Several computational and experimental approaches for detection of somatic retroelement insertions was developed in the past few years. These approaches were successfully applied to detect somatic insertions in clonally expanded tumor cells. At the same time, identification of somatic insertions presented in small proportion of cells, such as neurons, remains a considerable challenge. RESULTS: In this study, we developed a normalization procedure for library enrichment by DNA sequences corresponding to rare somatic RE insertions. Two rounds of normalization increased the number of fragments adjacent to somatic REs in the sequenced sample by more than 26-fold, and the number of identified somatic REs was increased by 8-fold. CONCLUSIONS: The developed technique can be used in combination with vast majority of modern RE identification approaches and can dramatically increase their capacity to detect rare somatic RE insertions in different types of cells.

Quantitative profiling reveals minor changes of T cell receptor repertoire in response to subunit inactivated influenza vaccine.

Vaccination against influenza is widely used to protect against seasonal flu epidemic although its effectiveness is debated. Here we performed deep quantitative T cell receptor repertoire profiling in peripheral blood of a healthy volunteer in response to trivalent subunit influenza vaccine. We did not observe significant rebuilding of peripheral blood T cell receptors composition in response to vaccination. However, we found several clonotypes in memory T cell fraction that were undetectable before the vaccination and had a maximum concentration at day 45 after vaccine administration. These cells were found in lower concentration in the course of repertoire monitoring for two years period. Our observation suggests a potential for recruitment of only a limited number of new T cells after each seasonal influenza vaccination.

Most recent AluY insertions in human gene introns reduce the content of the primary transcripts in a cell type specific manner.

Being the most effectively transposed primate-specific SINEs, Alu elements are present in more than one million copies in the human genome and include most recently transposed subsets of AluY elements that are polymorphic in humans. Although Alu elements are commonly thought to play an essential role in shaping and functioning of primate genomes, the understanding of the impact of recent Alu insertions on human gene expression is far from being comprehensive. Here we compared hnRNA contents for allele pairs of genes heterozygous for AluY insertions in their introns in human cell lines of various origins. We demonstrated that some AluY insertions correlated with decreased content of the corresponding hnRNAs. The effect observed does not depend on sequences of Alu elements and their orientation but is likely to be cell type specific.

Whole-genome experimental identification of insertion/deletion polymorphisms of interspersed repeats by a new general approach.

A new experimental technique for genome-wide detection of integration sites of polymorphic retroelements (REs) is described. The technique allows one to reveal the absence of a retroelement in an individual genome provided that this retroelement is present in at least one of several other genomes under comparison. Since quite a number of genomes are compared simultaneously, the search for polymorphic REs insertions is very efficient. The technique includes two whole-genome selective PCR amplifications of sequences flanking REs: one for a particular genome and another one for a mixture of ten different genomes. A subsequent subtractive hybridization of the obtained amplicons with DNA of a particular genome as driver results in isolation of polymorphic insertions. The technique was successfully applied for identification of 41 new polymorphic human AluYa5/Ya8 insertions. Among them, 18 individual Alu elements first sequenced in this work were not found in the available human genome databases. This result suggests that significant part of polymorphic REs were not identified during genome sequencing and remain to be detected and characterized. The proposed method does not depend on preliminary knowledge of evolutionary history of retroelements and can be applied for identification of insertion/deletion polymorphic markers in genomes of different species.